Sparber et al. BMC Medical Genetics 2020, 21(Suppl 1):197
https://doi.org/10.1186/s12881-020-01128-5

CASE REPORTOpen Access

Recessive myotonia congenita caused by ahomozygous splice site variant in CLCN1
gene: a case reportPeter Sparber* , Margarita Sharova, Alexandra Filatova, Olga Shchagina, Evgeniya Ivanova, Elena Dadali and
Mikhail Skoblov
From 11th International Young Scientists School “Systems Biology and Bioinformatics” – SBB-2019
Novosibirsk, Russia. 24-28 June 2019

Abstract
Background: Myotonia congenita is a rare neuromuscular disease, which is characterized by a delay in muscle
relaxation after evoked or voluntary contraction. Myotonia congenita can be inherited in a dominant (Thomsen
disease) and recessive form (Becker disease) and both are caused by pathogenic variants in the CLCN1 gene.
Noncanonical splice site variants are often classified as variants of uncertain significance, due to insufficient accuracyof splice-predicting tools. Functional analysis using minigene plasmids is widely used in such cases. Moreover,functional analysis is very useful in investigation of the disease pathogenesis, which is necessary for development of
future therapeutic approaches. To our knowledge only one noncanonical splice site variant in the CLCN1 gene wasfunctionally characterized to date. We further contribute to this field by evaluation the molecular mechanism of
splicing alteration caused by the c.1582 + 5G > A in a homozygous state.
Case presentation: We report a clinical case of an affected 6-y.o boy with athletic appearance due to muscle
hypertrophy, calf muscle stiffness, cramping and various myotonic signs in a consanguineous family with no historyof neuromuscular disorders. The neurological examination showed percussion-activated myotonia in the hands andlegs. Plasma creatine kinase enzyme and transaminases levels were normal. Electromyography at the time of examinationshows myotonic runs in the upper and lower extremities.
Conclusions: Functional analysis of the variant in a minigene system showed alteration of splicing leading to loss offunction, thereby confirming that the variant is pathogenic.
Keywords: Myotonia congenita, Becker disease, Functional analysis, Splicing, Case report* Correspondence: psparber93@gmail.com
Research Centre for Medical Genetics Moskvorechie 1, Moscow 115522,
Russia© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Sparber et al. BMC Medical Genetics 2020, 21(Suppl 1):197

Background
Myotonia congenita (MC) belongs to the group of nondystrophic myotonias along with paramyotonia andsodium channel myotonia. Clinically myotonia definedby muscle stiffness in various muscle groups, percussion
myotonia, and the “warm-up” phenomenon (relieve in
myotonia after repetitive contractions) with myotonic
discharges seen on electromyography (EMG) [1]. MC is
associated with pathogenic variants in the CLCN1 gene,which encode for the skeletal muscle chloride channel
CLC-1 and can be inherited in a dominant or recessive
trait. The recessive form of MC (Becker disease –OMIM #255700) typically has a more severe phenotype
and is considered to be more widespread than the dominant one (Thomsen disease – OMIM #160800) [2].
The overall prevalence of MC is 1:100′000 according
to the Orphanet database [3]. However the prevalence of
MC can be as high as 1:10′000 in Northern Finland [4].
The disease onset of recessive MC is usually in the firstor the second decade of life starting with painless myotonia in the lower limbs and progresses to the arms, facial muscles and neck. Patients can have an athletic
appearance due to muscle hypertrophy. Transient muscular weakness is also a common feature.
We are reporting a clinical case of a Turkmen consanguineous family where a 6-y.o boy at the time of examination had calf muscle stiffness with periodic muscle
spasms and various myotonic phenomena’s without
signs of muscle dystrophy. Sanger sequencing of the
CLCN1 gene identified a homozygous variant at the intron 14 - c.1582 + 5G > A. Functional analysis in a minigene system was performed in order to evaluate theimpact of the variant on splicing.
Case presentation
A family of Turkmen origin was referred to genetic consultation for progeny prediction. One of their three children - a 6-year old boy was examined due to calf muscle
stiffness and cramping in various muscle groups. The
proband was born in consanguineous marriage - his parents were third cousins. Gestation and delivery were uneventful. He developed normally for the first year of life
when painless muscle stiffness of the calf muscle was
first noticed. The parents had two healthy daughters
without any neurological signs. No history of neuromuscular disorders was present in the family.
Upon examination at the age of six, the proband had
an athletic appearance due to muscle hypertrophy. The
neurological examination showed percussion-activatedmyotonia in the hands and legs. After a maximum voluntary contraction of the hand, the proband could not
fully open his fist, but repeated attempts improve myotonia due to the warm-up phenomenon. Plasma creatine
kinase enzyme and transaminases levels were normal.

Page 2 of 5

Electromyography shows myotonic runs in biceps brachii, vastus lateralis and tibialis anterior muscles.
Based on the clinical findings a recessive form of MC
was suspected. Sanger sequencing of all CLCN1 exonsand the nearest intron regions was performed. It revealed a homozygous variant in the intron 14 of the
CLCN1 gene - c.1582 + 5G > A. Following segregation
analysis in the family showed that both parents are heterozygous for this variant (Fig.1).
Homozygous or compound heterozygous pathogenic
variants in the CLCN1 gene lead to recessive MC also
known as Becker disease. c.1582 + 5G > A variant was
described as pathogenic in a previous work [5].
However, no functional studies were made to confirm the functional consequences of the discovered
variant and according to ACMG guidelines this variant should be classified as a “variant of uncertain
significances” (VUS) [6]. This means that we cannot
be sure about the pathogenic role of the describedvariant and his causality. In silico analysis using
Human Splicing finder 3.1 [7] predicted a broken
wild-type donor site.
In order to determine the impact of the described variant on splicing, we performed a minigene-splicing assay.
We cloned the entire 14 exon with the flanking introniсsequences containing the variant of interest into pSpl3Flu plasmid [8] and transfected HEK293T cells. Subsequent RT-PCR analysis with plasmid-specific primers
showed that the presence of c.1582 + 5G > A variant leadto two independent pathogenic splicing alterations(Fig. 2). Sanger sequencing of RT-PCR products revealed
that c.1582 + 5G > A variant disrupt the donor splice site(SS) of intron 14, which leads to activation of two upstream exonic cryptic SS. The activation of the first
cryptic SS causes a 57-nucleotide truncation of exon 14(Δ57) resulted in an in-frame deletion of 19 amino acids(p.(Gly509_ Ile527del)). These 19 amino acids encode
for part of the fifteenth transmembrane α-helices of CLC1 channel (helix O) and other pathogenic missense variants were described in this exon leading to MC [9, 10].
The second pathogenic splicing event is a 91-nucleotide
truncation of exon 14 (Δ91) that leads to a frame-shift(p.(Val498GlufsTer5)) and a formation of a prematurestop codon (PTC). Such mRNA isoform should be degraded by nonsense-mediated decay (NMD) [11]. According to functional investigation missense variants in exon
14 of the CLCN1 gene are considered to be similar to lossof function (LoF) variants based on their effect on channel
conductance [12]. This means that in our case both splicing alterations are predicted to cause LoF of CLC-1 protein. Thereby after performing functional study according
to the ACMG guidelines we reclassify c.1582 + 5G > A
variant as a class 4 – likely pathogenic (criteria PM2, PP3
and PS3).

Sparber et al. BMC Medical Genetics 2020, 21(Suppl 1):197

Page 3 of 5

Fig. 1 Left panel shows the pedigree of the affected family. Black symbol indicate affected individual. Black dotes indicated unaffected carriers –the probands parents and the fetus. Right panel shows the electropherograms of the identified variant in the proband and his parents

Fig. 2 Functional analysis results. The minigene assay revealed that c.1582 + 5G > A variant leads to two independent pathogenic splicing
alteration: a 57 (Δ57) and a 91 (Δ91) nucleotide truncation. On the top schematic minigene design is depicted. WT- wild type, Mut- mutation. The
uncropped version of the gel image is available as additional file 1

Sparber et al. BMC Medical Genetics 2020, 21(Suppl 1):197

Discussion and conclusions
CLC-1 is the most abundant chloride channel expressed
predominantly on the sarcolemma of skeletal muscles
and plays a key role in excitability regulation. In humanmuscles, up to 80% of the resting membrane conductance is contributed by chloride conductance [13]. The
chloride channels in muscle cells are activated after anaction potential and are necessary for restoring the resting membrane potential. If the chloride channels fail toactivate, K+ starts to accumulate in the extracellularspace, which leads to membrane depolarization that at a
certain level can initiate self-activation resulting in a
prolonged muscle constriction that is observed in myotonia patients [14].
To date 280 pathogenic variants in CLCN1 are described according to HGMD professional database and include missense, nonsense, splicing substitutions, andframeshift variants. Different pathogenic variants can leadto different forms of MC. The recessive form of MC is associated with biallelic LoF variants that can be foundacross all gene body, except for truncation variants locatedclose to the C-terminus. Missense pathogenic variants canlead to both recessive and dominant MC. Dominant MС
is associated with missense variants, and truncating variants, escaping NMD leading to a dominant-negative effecton CLC-1 homodimer channel [2].
Because of the diversity in the molecular bases of MC,
it is rather difficult to establish the mode of inheritancebased on the patient’s genotype and only functional investigations can resolve this issue. Moreover, functional
studies increase our knowledge in understanding thepathogenesis of MC, which is crucial for more precise
genetic counseling and developing future therapeutic
approaches. Aberrant splicing is known to cause MC,
however, it is very difficult to interpret variants that lay
outside of the canonical SS dinucleotide. These intronic
variants are often classified as VUS and evaluating theirfunctional consequences is very important especially forfamilies that consider procreation.
The most informative results for splicing analysis are
obtained by using RT-PCR analysis from a diseaserelevant tissue. Unfortunately, in many cases, it is notpossible to perform such analysis for many reasons including medical and ethical issues. Nevertheless, several
publications including the work of van der Klift et al.
[15] showed that minigenes assays are highly concordant
with patient RNA analysis in terms of rather or not thevariant is pathogenic. In our case, the minigene assay revealed complete absence of the WT transcript and thepresence of two aberrant isoforms. Further analysis of
the mutated isoforms and their effect on the protein
level led us to the conclusion that both transcripts
should lead to a LoF which is the main pathogenic
mechanism in recessive MC.

Page 4 of 5

Here we present a clinical report of a proband with recessive form of MС in a consanguineous family with a variant
affecting splicing in a homozygous state. This variant wasreported as pathogenic before, however, no functional conformation was performed. Due to the fact that the proband
mother was pregnant, we decided to functionally investigate
this variant for performing proper prenatal diagnosis. By
splicing minigene assay, we confirmed the pathogenic roleof the splicing variant on the molecular level. At 10–11
weeks of gestation, a prenatal diagnosis was performed
from chorion villi samples showing that the fetus is a heterozygous carrier of c.1582 + 5G > A variant.

Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s12881-020-01128-5.
Additional file 1. Uncropped version of the gel image in Fig. 2

Abbreviations
MC: Myotonia congenital; CLCN1: Chloride voltage-gated channel 1;
LoF: Loss of function; VUS: Variant of uncertain significances;
EMG: Electromyography; ACMG: American college of medical genetics;
NMD: Nonsense-mediated decay; HGMD: Human gene mutation database;
HGVS: Human genome variation society; OMIM: Online mendelian
inheritance in man
Acknowledgements
Not applicable.
About this supplement
This article has been published as part of BMC Medical Genetics Volume 21
Supplement 1, 2020: Selected Topics in “Systems Biology and Bioinformatics”- 2019: medical genetics. The full contents of the supplement are available
online at https://bmcmedgenet.biomedcentral.com/articles/supplements/
volume-21-supplement-1.
Authors’ contributions
PS performed the functional analysis and wrote the manuscript. MS –performed the functional analysis. AF-wrote and revised the manuscript. OS– analysed the Sanger sequencing data. EI- performed the PCR and Sanger
sequinning of the CLCN1 gene. ED – performed the clinical examination of
the patient. MS – wrote and revised the manuscript. All authors read andapproved the final manuscript.
Funding
Publication costs have been funded by Ministry of Science and Higher
Education of the Russian Federation. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of
the manuscript.
Availability of data and materials
The datasets used and analysed during the current study are available fromthe corresponding author upon reasonable request. c.1582 + 5G > A variant
was submitted to LOVD database. Variant ID #0000598290.
Ethics approval and consent to participate
This study was approved by the Institutional Review Board of the ResearchCentre for Medical Genetics, Russia and was performed in accordance with
the Declaration of Helsinki. For this work written informed consent was
obtained from all participants. For participants under 16 years old written
informed consent was obtained from the participant’s parent.

Sparber et al. BMC Medical Genetics 2020, 21(Suppl 1):197

Consent for publication
For this work written informed consent was obtained from all participants
for the clinical examination and the publication of their anonymized dataand publication of identifying images. For participants under 16 years old
written informed consent was obtained from the participant’s parent. No
identifying images were used in this work.
Competing interests
The authors declare that they have no competing interests.
Published: 22 October 2020
References
1. Sansone, V.A., The Dystrophic and Nondystrophic Myotonias. Continuum(Minneap Minn), 2016. 22(6, Muscle and Neuromuscular Junction Disorders):
p. 1889-1915.
2. Fialho D, et al. Chloride channel myotonia: exon 8 hot-spot for dominantnegative interactions. Brain. 2007;130(Pt 12):3265–74.
3. Orphanet Report Series - Prevalence of rare diseases: Bibliographic data - June2018 - Number 1.
4. Papponen H, et al. Founder mutations and the high prevalence of
myotonia congenita in northern Finland. Neurology. 1999;53(2):297–302.
5. Ivanova EA, et al. The spectrum of CLCN1 gene mutations in patients withnondystrophic Thomsen's and Becker's myotonias. Genetika. 2012;48(9):1113–23.
6. Richards S, et al. Standards and guidelines for the interpretation ofsequence variants: a joint consensus recommendation of the American
College of Medical Genetics and Genomics and the Association for
Molecular Pathology. Genet Med. 2015;17(5):405–24.
7. Desmet FO, et al. Human splicing finder: an online bioinformatics tool topredict splicing signals. Nucleic Acids Res. 2009;37(9):e67.
8. Filatova AY, et al. Functional reassessment of PAX6 single nucleotide
variants by in vitro splicing assay. Eur J Hum Genet. 2019;27(3):488–93.
9. Moon IS, et al. Novel CLCN1 mutations and clinical features of Koreanpatients with myotonia congenita. J Korean Med Sci. 2009;24(6):1038–44.
10. Tincheva S, et al. Myotonia congenita type Becker in Bulgaria: first
genetically proven cases and mutation screening of two presumableendemic regions. Neuromuscul Disord. 2016;26(10):675–80.
11. Pereverzev AP, et al. Method for quantitative analysis of nonsense-mediatedmRNA decay at the single cell level. Sci Rep. 2015;5:7729.
12. Ha K, et al. Electrophysiological characteristics of six mutations in hClC-1 of
Korean patients with myotonia congenita. Mol Cells. 2014;37(3):202–12.
13. Bryant SH, Morales-Aguilera A. Chloride conductance in normal andmyotonic muscle fibres and the action of monocarboxylic aromatic
acids. J Physiol. 1971;219(2):367–83.
14. Tang CY, Chen TY. Physiology and pathophysiology of CLC-1: mechanismsof a chloride channel disease, myotonia. J Biomed Biotechnol. 2011;2011:
685328.
15. van der Klift HM, et al. Splicing analysis for exonic and intronic mismatch
repair gene variants associated with lynch syndrome confirms highconcordance between minigene assays and patient RNA analyses. Mol
Genet Genomic Med. 2015;3(4):327–45.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

Page 5 of 5